TACC3 is a microtubule plus end-tracking protein that promotes axon elongation and also regulates microtubule plus end dynamics in multiple embryonic cell types.
Bottom Line: Using high-resolution live-imaging data on tagged +TIPs, we show that TACC3 localizes to the extreme microtubule plus end, where it lies distal to the microtubule polymerization marker EB1 and directly overlaps with the microtubule polymerase XMAP215.TACC3 also plays a role in regulating XMAP215 stability and localizing XMAP215 to microtubule plus ends.Taken together, our results implicate TACC3 as a +TIP that functions with XMAP215 to regulate microtubule plus end dynamics.
Affiliation: Department of Biology, Boston College, Chestnut Hill, MA 02467.Show MeSH
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Mentions: To determine the plus end tracking mechanism of TACC3, we constructed several structural domain mutants of TACC3 (Figure 5A). Full-length GFP-TACC3 accumulates at all growing MT plus ends, as designated by mKate2-EB1 comets (Figure 5B and Figure 5 Supplemental Movie 1). Deleting the highly conserved N-terminal domain did not prevent plus end tracking (Figure 5, E–G, and Figure 5 Supplemental Movie 2). However, it did result in longer GFP-TACC3 plus end comets compared with controls, as well as longer EB1-GFP comets (Figure 5, E and F, and Figure 5 Supplemental Movie 2). These data suggest that the conserved N-terminal domain is involved in restricting TACC3 to the extreme plus end and also that TACC3 localization may affect EB1 binding to MT plus ends as well. Conversely, removing either the entire TACC domain (unpublished data) or even just the second coiled-coil domain of the TACC domain completely abrogated MT plus end tracking (Figure 5, H–J, and Figure 5 Supplemental Movie 3), demonstrating that the TACC domain, and in particular the second coiled-coil domain, is essential for TACC3 plus end tracking. We then observed significant interference with plus end tracking ability when TACC3 was tagged with GFP on the C-terminal end (Figure 5, K–M, and Figure 5 Supplemental Movie 4), further supporting the importance of the TACC domain for plus end tracking. However, whereas the TACC domain is necessary for promoting plus end tracking, it is not sufficient, as the entire TACC domain alone did not localize to MT plus ends (Figure 5, N–P, and Figure 5 Supplemental Movie 4).
Affiliation: Department of Biology, Boston College, Chestnut Hill, MA 02467.